The long-term objective of this proposal is to understand the diverse functions of chromatin in the nucleus. Chromatin in the eukaryotic nucleus is multidimensional. There are covalent modifications of the histones, histone variants, ATP-driven chromatin remodeling factors, non- histone chromosomal proteins, and non-nucleosomal chromatin particles. Notably, each of these dimensions of chromatin affects gene expression. This proposal is focused on the analysis of non-nucleosomal particles. Canonical nucleosomes have been extensively studied and are very well understood. In contrast, little is known about non-nucleosomal chromatin particles, which are a dynamic component of chromatin. One example of a non-nucleosomal chromatin particle is the prenucleosome, which functions as a precursor to the nucleosome in the assembly of chromatin. Yet, although prenucleosomes were identified in the study of chromatin assembly, prenucleosomes or prenucleosome-like particles might be generated wherever nucleosomes are disrupted or altered, such as at promoters and enhancers. The proposed work is designed to gain a better understanding of non-canonical chromatin particles with particular attention to their functions in chromatin dynamics and gene expression. Specific Aim 1 will examine the structure and properties of prenucleosomes by using purified and defined components. New advances have been made that will allow the analysis of prenucleosome structure, prenucleosome mobility, the conversion of prenucleosomes to nucleosomes, and the effect of prenucleosomes on elongating RNA polymerase II. Specific Aim 2 is directed toward the development of new methods and approaches to the study of a special class of native non-nucleosomal chromatin particles that contain all four core histones and are found at the promoter regions of active genes. These new techniques will be used to investigate the occurrence of the non-nucleosomal particles in different biological contexts as well as for the purification and biochemical characterization of these particles. The relation between the biochemically identified prenucleosomes and the native non-nucleosomal particles in cells will also be investigated. The proposed work will provide new insights into the structure, composition, properties, functions, and occurrence of non-canonical chromatin particles and their roles in chromatin dynamics and gene expression.